Optimizing locked nucleic acid/2’-O-methyl-RNA fluorescence in situ hybridization (LNA/2’OMe-FISH) procedure for bacterial detection

Abstract

Despite the successful application of LNA/2’OMe-FISH procedures for bacteria detection, there is a lack of knowledge on the properties that affect hybridization. Such information is crucial for the rational design of protocols. Hence, this work aimed to evaluate the effect of three essential factors on the LNA/2’OMe hybridization step—hybridization temperature, NaCl concentration and type and concentration of denaturant (formamide, ethylene carbonate and urea). This optimization was performed for 3 Gram-negative bacteria ( Escherichia coli , Pseudomonas aeruginosa and Citrobacter freundii ) and 2 Gram-positive bacteria ( Enterococcus faecalis and Staphylococcus epidermidis ), employing the response surface methodology and a Eubacteria probe. In general, it was observed that a high NaCl concentration is beneficial (from 2 M to 5 M), regardless of the denaturant used. Urea, formamide and ethylene carbonate are suitable denaturants for LNA/2’OMe-FISH applications; but urea provides higher fluorescence intensities among the different bacteria, especially for gram-positive bacteria and for P . aeruginosa . However, a unique optimal protocol was not found for all tested bacteria. Despite this, the results indicate that a hybridization solution with 2 M of urea and 4 M of NaCl would be a proper starting point. Furthermore, a hybridization temperature around 62°C, for 14 bp probes with LNA monomers at every third position of 2′OMe and 64% of GC content, should be use in initial optimization of new LNA/2’OMe-FISH protocols.

Can Vitamin B12 Assist the Internalization of Antisense LNA Oligonucleotides into Bacteria?

“…In this study, two different kinds of LNA antisense oligonucleotides were designed and synthesized to target the acpP gene in E. coli ( Figure 2 a): an LNA/DNA gapmer (ASO gapmer ) and an LNA/2′OMe steric blocker (ASO steric ) [ 28 , 29 ]. While steric blockers have been widely tested in bacteria [ 4 , 27 , 30 , 31 , 32 ], gapmers have been mostly studied in mammalian cells [ 11 ].…”

Lipoplexes to Deliver Oligonucleotides in Gram-Positive and Gram-Negative Bacteria: Towards Treatment of Blood Infections

“…FISH was then applied. Traditional in vitro FISH starts with a permeabilization/fixation step, needed to render the bacterial envelope permeable to the ONs, based on pretreating the bacteria with chemicals that are toxic for in vivo application [ 35 , 36 , 37 ]. In this study, FISH was applied without this pretreatment, based on Santos et al with slight modifications [ 19 ].…”

Friends with Benefits: An Inside Look of Periodontal Microbes’ Interactions Using Fluorescence In Situ Hybridization—Scoping Review

“…Fluorescence in situ hybridization (FISH) has proven to be particularly useful to describe the microbial composition and spatial organization of mixed microbial infections, in time and space within their natural context. This molecular technique relies on the hybridization of single-stranded, fluorescently labeled DNA-, RNA-, or nucleic acid mimics-targeted oligonucleotides probes with fluorescent molecules (e.g., fluorochromes) that hybridize to its complementary conserved 16S or 23S rRNA sequences in the microorganism [ 10 , 11 , 12 ].…”